5α-dihydrotestosterone reduces renal Cyp24a1 expression via suppression of progesterone receptor

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   Figure 1

   Influence of 5α-dihydrotestosterone (DHT) on sex steroid receptors in kidney. After orchiectomy and recovery of mice for 2 weeks, male mice (n = 5 or 6 per each group) were treated with vehicle alone (vehicle), with DHT (50 μg/day) or DHT plus bicalutamide (1 mg/day) for 5 days, and Kap (A), Ar (B), Esr1 (C) and Pgr (D) mRNA levels were determined by quantitative RT-PCR. Gapdh mRNA was used as in internal control. Values represent means ± s.e.m. *P < 0.05 vs orchiectomy vehicle-alone (control) treated group. ^# P < 0.05 vs DHT-bicalutamide-treated group.
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   Figure 2

   Influence of 5α-dihydrotestosterone (DHT) on expression of sex steroid receptors in human hormone-dependent breast cancer cell. T47D or MCF-7 cells were treated with vehicle alone or with DHT, and AR (A and D), ESR1 (B and E), PGR (C and F) mRNA levels were determined by quantitative RT-PCR. Cells were grown in phenol red-free DMEM/F12 medium supplemented with 2% charcoal-treated FBS for 5 days before daily treatments with DHT at 10 nM. GAPDH mRNA was used as in internal control. Values represent means ± s.e.m. of three separate experiments, P < 0.05 vs vehicle-alone (control)-treated group.
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   Figure 3

   Impact of siRNA-mediated knockdown or PGR overexpression on the Cyp24a1 and Vdr mRNA expression. PCT cells were transfected with either 100 nM control siRNA (con si) or progesterone receptor siRNA (Pgr si), and then cultured in medium containing 2% FBS for a further 24 h (A). Cyp24a1 (B), and Vdr (C) mRNA levels were determined by quantitative RT-PCR, using Gapdh mRNA as an internal control. The values represent means ± s.e.m. of four experiments. *P < 0.05 when PGR siRNA treated PCT cells are compared to control siRNA treated PCT cells. PCT cells were transfected with PGRa or PGRb expression vectors, and then cultured in medium containing 2% FBS for a further 24 h (D, m = mouse, h = human). Cyp24a1 (E), and Vdr (F) mRNA levels were determined by quantitative RT-PCR, using Gapdh mRNA as an internal control. The values represent means ± s.e.m. of four experiments. *P < 0.05 when PGRa or PGRb expression vectors treated PCT cells are compared to control vector treated PCT cells.
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   Figure 4

   Standard ChIP qPCR analysis of PGR occupancy on Cyp24a1 in the mouse PCT cell line. After PCT cells were cultured in medium containing 2% FBS for a further 24 h, they were treated with 10 nM progesterone for 1 h, and then crosslinked by formaldehyde. The region of PGR occupancy on Cyp24a1 gene is located 7775 bp upstream the transcription initiation site (170,322,016 in chromosome 2). The full palindromic PRE motif was indicated with red box (A). Pgr, Ppargc1a, and Sox17 gene promoter were introduced with positive control (B and C). A full color version of this figure is available at http://dx.doi.org/10.1530/JME-17-0187.
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   Figure 5

   Influence of 5α-dihydrotestosterone (DHT) on vitamin D-related genes. After orchiectomy and recovery of mice for 2 weeks, male mice were treated with vehicle alone (vehicle), with DHT (50 μg/day) or DHT plus Bicalutamide (1 mg/day) for 5 days, and renal Cyp24a1 (A), Vdr (B), and Cyp27b1 (C) mRNA levels were determined by quantitative RT-PCR. Gapdh mRNA was used as in internal control. Values represent means ± s.e.m. ^§ P < 0.05 vs sham control group. *P < 0.05 vs orchiectomy vehicle-alone (control) treated group. ^# P < 0.05 vs DHT-bicalutamide treated group. In panel (D), the levels of renal CYP24A1, and VDR compared to PRa expression were analyzed by Western blotting. For these experiments, 40 μg samples of protein extracts were analyzed, and β-ACTIN were also examined as internal controls. Relative amounts of protein expression in orchiectomy vs sham control was evaluated based on Western blot analysis. PCT cells were treated with vehicle alone (control) or with DHT, and Pgr mRNA levels were determined by RT-PCR (E), and Cyp24a1 (F), and Vdr (G) mRNA levels were determined by quantitative RT-PCR. Cells were grown in phenol red-free DMEM/F12 medium supplemented with 2% charcoal-treated FBS for 5 days before daily treatments with DHT at 10 nM. Gapdh mRNA was used as in internal control. In panel (H), the levels of CYP24A1, and VDR in the corresponding PRa expression were assessed by Western blotting. For these experiments, 40 μg samples of protein extracts were analyzed, and β-ACTIN were also examined as internal controls. The values represent means ± s.e.m. of four experiments. *P < 0.05 when DHT-treated PCT cells are compared to vehicle-treated PCT cells.
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   Figure 6

   Influence of DHT to vitamin D and vitamin D-related genes expression. Level of 25-hydroxyvitamin D in mouse serum is determined by enzyme-linked immunosorbent assay (A). Values represent means ± s.e.m. *P < 0.05 vs orchiectomy vehicle-alone (control) treated group. ^# P < 0.05 vs DHT-bicalutamide treated group. Schematic representation highlighting the molecular link between androgen (DHT), progesterone receptor, CYP24A1, and the vitamin D metabolites (B). Cabp9k and Cabp28k mRNA levels were determined by quantitative RT-PCR (C and D). Gapdh mRNA was used as in internal control. Values represent means ± s.e.m. *P < 0.05 vs sham control group.

• © 2018 Society for Endocrinology